New scanning electron microscopic method for determination of adipocyte size in humans and mice

Objective: To evaluate a new scanning electronic microscopic (EM) method for assessing fat cell sizes and compare fat cell size distribution in human adipose tissue from different fat depots before and after weight loss. Research Methods and Procedures: Identical human fat tissue biopsies were separated into two fractions: one used to prepare a fat cell suspension by collagenase digestion followed by photomicrography (collagenase method) and the other fixed in formalin for EM analysis. The EM method was evaluated further by determining fat cell sizes from lean and ob/ob mice. Finally, the EM method was used to assess fat cell sizes in biopsies of different human depots from before and after weight loss. Results: Fat cell size distributions measured by the two methods were not identical, but differences were generally small. The EM method reproduced the well‐documented fat cell size difference between lean and ob/ob mice. Large variation was detected in fat cell distributions among three depots in humans. Weight loss reduced fat cell sizes in subjects with large baseline fat cells but had no effect in subjects with small baseline fat cell sizes. Discussion: Our results suggest that the EM method may be a useful alternative for fat cell size analysis of clinical samples.     

Ultrastructure of the nephridio-circulatory connections in Tubulanus annulatus (Nemertini,Anopla)

Summary The protonephridial terminal organs in the nemertean Tubulanus annulatus form an integral part of the blood vessel wall. Both endothelial and muscle-cell layers of the vessel's wall are discontinued at the site of each terminal organ. The terminal organs are usually composed of from one to three terminal cells enclosing a central lumen provided with many microvilli and separated from the blood vessel's lumen by a membranous filtration area. The latter is perforated by numerous winding clefts formed by interdigitation of minute cytoplasmic pedicels arising from processes issued by each of the involved terminal cells. Ultrafiltration of blood plasma takes place across a filtration membrane which spans the cleft system and the basal lamina of the terminal cells. Fluid is propelled into the lumen of the terminal organs through the activity of ciliary bundles, one for each terminal cell involved, perhaps supplemented by vascular turgor. All efferent conduits of the protonephridium have profuse infoldings of the luminal cell surfaces and/or numerous pinocytotic pits suggestive of reabsorption of substances from the primary urine.Abbreviations BL basal lamina - C cilium - CP coated pit - CT collecting tubule - CV inzcoated vesicle - D dictyosome - E endothelial cell - F fenestration of endothelial cell - FA filtration area - FM filtration membrane - G glycogen granule - LV lateral vessel - M mitochondrion - MC muscle cell - MV microvillus - N nucleus of terminal cell - NE nucleus of endothelial cell - NP nephridiopore - PC protonephridial capillary cell - PT protonephridial tubule - R rootlet - TC terminal cell     

Cell lines from diamondback moth exhibiting differential susceptibility to baculovirus infection and expressing midgut genes

Abstract Six new cell lines were established from embryonic tissues of the diamondback moth, Plutella xylostella (L.). The cell lines showed differential characteristics, including growth in attachment or in suspension, susceptibility to a baculovirus infection and expression of genes involved in the glucosinolate detoxification pathway in R xylostella larvae. Five of the cell lines grew attached to the culture flask and one cell line grew unattached as a suspension cell line. The cell lines had population doubling times ranging from IS to 23 h. Among five of the P. xylostella cell lines examined for infection of a nucleopolyhe. drovirus from Autographa californica, AcMNPV four cell lines were highly susceptible to AcMNPV infection, but one was only semi-permissive to AcMNPV infection. The production of two recombinant proteins, a β-galactosidase of bacterial origin and a secreted alkaline phosphatase of eukaryotic origin, in the R xylostella cell lines was examined in comparison with that in the cell line Sf9 which is commonly used for recombinant protein production. In the P. xylostella cell lines, expression of three important midgut genes involved in the glucosinolate detoxification pathway, including the glucosinolate sulfatase genes GSS1 and GSS2 and the sulfatase modifying factor gene SUMF1、was detected. The R xylostella cell lines developed in this study could be useful in in vitro research systems for studying insec-virus interactions and complex molecular mechanisms in glucosinolate detoxification and insect-plant interactions.     

Sequential activation and loss of the pre-B cell Thy-1 gene in T-cell x pre-B cell somatic hybrids

In somatic cell hybrids between the pseudodiploid Thy-1^– Abelson-leukemia-virus-induced pre-B cell lymphoma RAW 253.1 and the Thy-1⁺ T-cell lymphoma, AKR1 (Thy-1⁺), all cells express the Thy-1 allele of the T-cell parent but most hybrid cells do not express the Thy-1 allele of the pre-B cell lymphoma parent. The Thy-1 allele of the pre-B cell parent, however, is spontaneously activated in a minor proportion of hybrid cells. By sorting for cells expressing the Thy-1 allele of the pre-B cell parent, derivative clones in which 100% of cells express both parental Thy-1 alleles can be isolated. Revertants with a phenotype identical with that of the original hybrid cell line can be isolated from these derivatives by sorting for nonexpression of the Thy-1 allele of the pre-B cell parent. These first-generation revertant cell lines have lost one copy of the Thy-1 gene derived from the pre-B cell lymphoma parent. By a further cycle of sorting, derivatives in which 100% of cells express both parental Thy-1 alleles can again be obtained. Second-generation revertants isolated by sorting these Thy-1⁺ hybrid cells for nonexpression of the Thy-1 allele of the pre-B cell parent no longer contain a normal copy of the pre-B cell Thy-1 allele and this surface antigen is no longer expressed by any cells in the population. These results are consistent with a mechanism that sequentially activates each copy of the Thy-1 gene derived from the pre-B cell lymphoma parent. Hybrids between the class D Thy-1 ^– mutant, AKR1(Thy-1^–d), in which the 5 region of the Thy-1 structural gene has been deleted, and RAW 253.1 cannot be activated to express either Thy-1 allele. This result indicates that a sequence upstream of exon 2 of the active Thy-1 allele is critical for the initial activation event.     

ISOLATION AND CHARACTERIZATION OF A CELL WALL‐DEFECTIVE MUTANT OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

Cell wall–defective strains of Chlamydomonas have played an important role in the development of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the distantly related homothallic species, C. monoica, we used UV mutagenesis to obtain a mutant strain with a defective cell wall. The mutant, cw‐1, was first identified on the basis of irregular colony shape and was subsequently shown to have reduced plating efficiency and increased sensitivity to lysis by a non‐ionic detergent as compared with wild‐type cells. Tetrad analysis of crosses involving the cw‐1 mutant confirmed 2:2 segregation of the cw:cw⁺ phenotypes, indicating that the wall defect resulted from mutation of a single nuclear gene. The phenotype showed incomplete penetrance and variable expressivity. Although some cells had apparently normal cell walls as viewed by TEM, many cells of the cw‐1 strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw‐1 isolates obtained from crosses involving the original mutant strain showed a marked enhancement of the mutant phenotype and may prove especially useful for future work involving somatic cell fusions or development of transformation protocols.     

A New Variant for Mathematical Analysis of Pulse Cytophotometric Data

The staining of DNA by specific fluorochromes provides a suitable method of receiving histograms in a short time by means of pulse cytometry. They represent the proliferative structure of cell populations at a high degree of statistical security. A method for quantitative determination of cell cycle phases (G_1-, S- and G₂ + M-phase) is presented which includes the fraction of cell debris in the calculation procedure. The advantages of this method are the elimination of overlapping between the fraction of debris and cell cycle phases and the quantitative determination of the fraction of cell debris offers the opportunity to get information on cytolytic potencies. Apart from the calculation of the various cell cycle phases the method provides criteria on the adaptation of mathematical analysis to primary data.     

Establishment and characterization of insect cell lines from 10 lepidopteran species

Summary Cell lines from selected lepidopteran species were established for the overall purpose of use in baculovirus production. A total of 36 new cell lines from 10 lepidopteran species were generated, including cell lines from a pyralid, the European corn borer,Ostrinia nubilalis, a plutellid, the diamondback moth,Plutella xylostella, as well as eight noctuids: the black cutworm,Agrotis ipsilon, the celery looper,Anagrapha falcifera, the velvetbean caterpillar,Anticarsia gemmatalis, the corn earworm,Helicoverpa zea, the tobacco budworm,Heliothis virescens, the beet armyworm,Spodoptera exigua, the fall armyworm,Spodoptera frugiperda, and the cabbage looper,Trichoplusia ni. Tissues used for cell line establishment included fat bodies, ovaries, testes, or whole embryos/larvae/pupae. All the cell lines were subcultured numerous times, characterized by isoenzyme analysis and/or deoxyribonucleic acid amplification fingerprinting using polymerase chain reaction, and stored in liquid nitrogen. Many of the cell lines were adapted to grow in serum-free medium, with cell lines fromA. ipsilon andH. virescens being adapted to suspension culture, using shaker flasks. The potential use for these cell lines in baculovirus production is discussed. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion sex, age, marital status, or handicap.     

Changes in glial fibrillary acidic protein and karyotype during culturing of two cell lines established from human glioblastoma multiforme

Summary Two human cell lines (GL15 and GL22) derived from glioblastoma multiforme were established and characterized by immunohistochemical and cytogenetic techniques. The expression of glial fibrillary acidic proteins and the karyotype were analyzed at different passages for both cell lines. The course of marker-pattern differed in the two cell lines. The main findings were a cell-density-dependent expression of glial fibrillary acidic protein in the cell line GL15 at all passages and a decreased expression of this protein over time in the cell line GL22. Both cell lines had hyperdiploid karyotypes and exhibited glioma-specific chromosomal abnormalities (gain of chromosome 7 and loss of chromosome 10). In the GL15 cell line no relevant chromosomal changes were produced during culturing, whereas in the GL22 cell line a hypodiploid clone appeared at the 42nd passage. The immunohistochemical and cytogenetic data resulting from this study confirm that the two cell lines established in our laboratory originated from astrocytic tumor cells.Abbreviations MHG malignant human gliomas - GFAP glial fibrillary acidic protein - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum - GTG banding trypsin-Giemsa banding - TBS TRIS-buffered saline 10 mM pH 7.6 - p short arm of chromosome; q long arm of chromosome - der derivative chromosome     

Tracing the ontogeny of stomatal clusters in Arabidopsis with molecular markers

The origin and process by which the mosaic of the different cell types is established during the development of the leaf epidermis in Arabidopsis are largely unknown, although the recent characterization of two mutants which develop stomatal clusters (four lips (flp) and too many mouths (tmm)) has opened up the possibility for genetic dissection of the stomata spacing. By using growth conditions which limit gas exchange with the open atmosphere, stomatal clusters that look like phenocopies of flp and tmm have been induced, suggesting that stomata spacing is under environmental as well as genetic control in Arabidopsis. The origin of these clusters has been addressed by following promoter activity for genes that are markers for competence for cell division (cdc2aAt), mitotic activity (cyc1aAt), and guard mother cell and developing guard cell identity (rha1). Their different expression patterns in the various cell types during epidermal differentiation and the asynchrony in the development of the various stomata that constitute each cluster suggest that these stomatal clusters derive from a single protodermal cell through a process that involves changes in cell fate in a subset of subsidiary cells. It was also found that guard cells express cdc2aAt and cyc1aAt, supporting the idea that they may remain competent for cell division.     

Altered matrix polysaccharides in cell walls of pocket galls formed by an aphid on Distylium racemosum leaves

The present work reports the results of a study on the isolation and characterization of matrix polysaccharides in the cell walls of galls formed by an aphid (Neothoracaphis yanonis) on Distylium racemosum leaves. Cell walls were isolated from both healthy Distylium leaf and gall tissues and then extracted sequentially with cyclohexane‐trans‐1,2‐diaminetetra‐acetate (CDTA), Na₂CO₃, 1 m KOH, and 4 m KOH. The amount of pectin solubilized from gall cell walls was approximately 2.6‐fold higher than the pectin solubilized from leaf cell walls, whereas the amount of hemicellulose solubilized from gall cell walls was 1.4‐fold higher than that from normal leaf cell walls. When the polysaccharides were fractionated by anion‐exchange chromatography, considerable increases in arabinose and galactose were observed in CDTA‐soluble pectic polymer (fraction PI‐1) from gall cell walls, whereas the gall cell walls had less xylose in 1 m KOH‐soluble hemicellulosic polymers (fractions HI‐2, HI‐3, and HI‐4) than did the cell walls from the healthy leaf. The hemicellulosic polymers of the gall cell walls exhibited distinctly different patterns of molecular mass, compared with the healthy leaf cell walls. These results suggest that an extensive change occurs in the matrix polysaccharide structure of the cell walls of Distylium galls formed by an aphid. In addition, many glycosylhydrolase activities were detected in the protein fraction solubilized with strong saline solution from the gall cell walls, and the activities of β‐galactosidase, β‐xylosidase and α‐l ‐arabinofuranosidase were considerably increased under gall formation.     

LIGHT AND ELECTRON MICROSCOPY OF GRAZING BY POTERIOOCHROMONAS MALHAMENSIS (CHRYSOPHYCEAE) ON A RANGE OF PHYTOPLANKTON TAXA1

Grazing of fluorescent latex beads, bacteria, and various species of phytoplankton by Poterioochromonas malhamensis (Pringsheim) Peterfi (about 8.0 μm in diameter) was surveyed. The alga ingested fluorescent beads and various live or killed and nomnotile or motile organisms including bacteria, blue-green algae, green algae, diatoms, and chrysomonads. The size range of grazed prey was from 0.1 to 6.0 μm for latex beads and from 1.0 μm (bacteria) to about 21 μm (Carteria inverse) for organisms. As many as 17 latex beads (2.0 μm) or more than 10 Microcystis cells (5–6 μm) were ingested by a single P. malhamensis cell. Following such grazing, the cell increased in volume by up to about 30-fold. The range of cell volume of ingested prey was from 0.52 μm³ (bacteria) to about 3178 μm³(Carteria inversa). This study demonstrates for the first time that P. malhamensis is capable of grazing algae 2–3 times larger in diameter than its own cell and of grazing intact motile algae. Poterioochromonas malhamensis is an omnivorous grazer. Food vacuole formation and digestion processes were examined. The membrane that was derived from the plasma membrane and surrounded the prey disappeared sometime after ingestion. The food vacuole was then formed by successive fusion of numerous homogeneous vesicles accumulated around the prey. The prey was enclosed in a single membrane-bound food vacuole and then digested.     

Cytotoxic Activity of Fungal Strains Isolated from the Ascidian Eudistoma vannamei

The cytotoxic activity at 50 μg/ml of extracts obtained from eleven fungal strains associated to Eudistoma vannamei, an endemic ascidian from Northeast Brazil, against two cell lines, i.e., the HCT‐8 (colon cancer) and the MDA‐MB‐435 (melanoma) cell lines, was investigated. The most promising extract (EV10) was obtained from a fungus identified as Aspergillus sp. by molecular analysis and was selected for bioassay‐guided isolation of its active principals. Large‐scale fermentation of EV10 in potato‐dextrose broth followed by chromatographic purification of the active extract from the liquid medium allowed the isolation of the isocoumarins mellein, cis‐4‐hydroxymellein, and trans‐4‐hydroxymellein, besides penicillic acid. All isolated compounds were tested for their cytotoxicity against the tumor cell lines MDA‐MB‐435 and HCT‐8 and revealed penicillic acid as the only cytotoxic compound (cell growth inhibitions >95%).     

COMPARATIVE PHYSIOLOGICAL STUDY OF MARINE DIATOMS AND DINOFLAGELLATES IN RELATION TO IRRADIANCE AND CELL SIZE. I. GROWTH UNDER CONTINUOUS LIGHT1,2

Cell division rates and chlorophyll a and protein contents for ten diatom and dinoflagellate species were measured. Species were chosen to include a wide range of cell size in terms of both cell volume and cell protein: from 0.004 ng protein/cell for a small Chaetoceros sp. to 2.2 ng protein/cell for Prorocentrum micans Ehrenberg. Experiments were conducted in batch or semi-continuous cultures at 21 C under continuous illumination from 8–256 μEin ^.m^-2'^.s^-1. Light saturation of cell division occurred at 32–80 μEin m^-1 s^-1 for all species, with no observable difference between the two phylogenetic groups. When the light-saturated cell division rates were plotted against cell size as protein/cell, the diatoms and dinoflagellates fell on two separate lines with the diatoms having higher rates. Chl a /protein ratios (μg/μg) decreased with increasing irradiance. The diatoms had higher chl a per unit protein. The relationship between cell division rate and the chl a/protein ratio is discussed.     

β-Glucosidase production by Trichoderma reesei QM9414 on wheat straw. Location and some kinetic features

In this paper we studied the conditions for the production of β-glucosidase from T. reesei QM9414 in batch cultures using milled and sieved wheat straw as sole carbon source. High β-glucosidase production in the presence of wheat straw, a more realistic substrate than commercial cellulose, was obtained. The influence of particle size of wheat straw on β-glucosidase production in cell-free, cell and cell-wall extracts was studied. The particle size of wheat straw notably influenced enzyme production in cell and extramycelial extracts but it was less important with respect to the cell wall bound enzyme. β-glucosidase production was studied along of the fermentation. The results suggest a close relation between β-glucosidase from cell extract and extramycelial broth; geneticin levels of inhibition of β-glucosidase biosynthesis in both fractions were similar, a fact that suggests a common origin for the enzyme. Kinetic parameters for β-glucosidase from cell free and cell extracts were V_max = 0.28 μmol/min/mg, K_M = 0.91 mM and V_max = 0.095 μmol/min/mg, K_M = 0.39 mM respectively. Kinetic parameters for β-glucosidase from cell-wall could not be calculated because experimental data did not fit the different monosubstrate equations.     

Immunological detection of the fungal parasite,Pythium sp.; the causative organism of red rot disease in Porphyra yezoensis

The red rot disease of Porphyra yezoensis Ueda (Rhodophyta) is caused by a parasitic fungus, Pythium sp. To facilitate the detection of this pathogen in infected thalli of P. yezoensis, polyclonal and monoclonal antibodies were prepared. Antibodies were raised against antigen prepared from an isolate of fungal hyphae obtained from red-rot infected thallus of P. yezoensis from Aichi Prefecture. Polyclonal antibody was obtained from the antisera of immunized rabbits. Monoclonal antibody was obtained from the culture supernatant of a hybridoma which had been established by cell fusion between a myeloma cell line and spleen cells of immunized mice. Hyphae were detected by means of indirect fluorescent antibody technique. Titers of polyclonal antibodies obtained were too low to recognize fungal hyphae that had penetrated the thalli of P. yezoensis; however, monoclonal antibody was useful for the detection of fungi that had penetrated algal thalli. The monoclonal antibody was specific for the Pythium sp. from red-rot infected thalli of P. yezoensis from Saga (western Japan) and from Aichi Prefectures (central Japan), but was ineffective for infections from Miyagi Prefecture (northern Japan). It is evident, therefore, that Pythium sp. can give rise to immunologically distinct groups of red rot disease. Based on chemical and enzymatic treatments, the antigenic determinant appeared to localize on the sugar chains of glycoconjugates or the polysaccharides of the hyphal cell wall.     

GALEIDINIIUM RUGATUM GEN. ET SP. NOV. (DINOPHYCEAE), A NEW COCCOID DINOFLAGELLATE WITH A DIATOM ENDOSYMBIONT1

A new sand‐dwelling dinoflagellate from Palau, Galeidinium rugatum Tamura et Horiguchi gen. et sp. nov., is described. The life cycle of this new alga consists of a dominant nonmotile phase and a brief motile phase. The motile cell transforms itself directly into the nonmotile cell after swimming for a short period, and cell division takes place in the nonmotile phase. The nonmotile cell possesses a dome‐like cell covering, which is wrinkled and equipped with a transverse groove on the surface. The cell has 10–20 chloroplasts and a distinct eyespot. The motile cell is Gymnodinium‐like in shape. The dinoflagellate possesses an endosymbiotic alga to which the chloroplasts belong and which is separated from the host (dinoflagellate) cytoplasm by a unit membrane. The endosymbiont cytoplasm also possesses its own eukaryotic nucleus and mitochondria. The eyespot is surrounded by triple membranes and is located in the host cytoplasm. Photosynthetic pigment analysis, using HPLC, revealed that G. rugatum possesses fucoxanthin as the principal accessory pigment instead of peridinin. The rbcL tree showed that G. rugatum is monophyletic with Durinskia baltica (Levander) Carty et Cox and Kryptoperidinium foliaceum (Stein) Lindemann and that this clade is closely related to the pennate diatom, Cylindrotheca sp. The endosymbiont of G. rugatum is therefore shown to be a diatom. Phylogenetic analysis based on small subunit rDNA sequences demonstrated that G. rugatum, D. baltica, and K. foliaceum, all of which are known to harbor an endosymbiont of diatom origin, are closely related.     

Production of Microbial Cells from Hydrocarbons

Hydrocarbon-assimilating yeasts and bacteria were isolated from soil and sewage. The optimal conditions of cell yield from liquid paraffine by a Torulopsis yeast and a Pseudomonas strain were studied. A Torulopsis yeast gave, in optimal condition, 70 percent cell yield on a weight conversion basis from light oil fraction. In a strain of Pseudomonas the additions of amino acids, Fe^{+ +}, Mg^{+ +} and Ca^{+ +} ions were effective for cell production. This strain showed, in optimal condition, 80 percent cell yield (wt%) from kerosene.     

Display of heterologous proteins on the surface of Lactococcus lactis using the H and W domain of PrtB from Lactobacillus delburueckii subsp. bulgaricus as an anchoring matrix

Aims: The aim of this study was to develop a cell‐surface display system for foreign antigens on the surface of a Lactococcus lactis strain using an H and W domain of PrtB from Lactobacillus delburueckii subsp. bulgaricus as an anchoring matrix. Methods and Results: To construct a cell‐surface display pACL1 vector, a derivative of pSECE1 vector, we amplified the H and W domain of the cell‐surface proteinase Prt B from Lact. bulgaricus using specific primers and then cloned it into a site downstream of the secretion signal sequence in the pSECE1 vector. The new system, designed for cell‐surface display of recombinant proteins on L. lactis, was evaluated by the expression and display of the FliC protein of Salmonella enterica serovar Enteritidis as a reporter gene (pALC1:FliC). The expression of the FliC protein by the transformed cells was analysed by Western blot analysis, and the localization of FliC on the cell surface was confirmed by immunofluorescence microscopy and flow cytometry analysis. A specific band corresponding in size (approx. 110 kDa) to FliC plus the anchor residues was detected by anti‐FliC antibody in the cell extract of L. lactis H61 harbouring pALC1:FliC, but not L. lactis H61 harbouring pALC1. In addition, flow cytometry and immunofluorescence microscopy revealed FliC‐specific positive signals and a significant increase of fluorescence, respectively, in cells harbouring pALC1:FliC compared with that in control cells harbouring the parental pALC1 plasmid. These findings demonstrated that FliC was successfully displayed on the cell surface by the anchor domain of PrtB. Conclusions: A pALC1 vector using the H and W domain of PrtB from Lact. bulgaricus as an anchoring matrix can be used to successfully display the FliC protein on the surface of L. lactis. Significance and Impact of the Study: This novel way of displaying heterologous proteins on the cell surface of L. lactis using the PrtB anchor domain should prove useful for the delivery of antigens and other proteins.